In vitro and in vivo studies have demonstrated that the biologically active vitamin D metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D3, calcitriol) suppresses proliferation and induces differentiation in various cell types, including human keratinocytes and melanocytes. Vitamin D is synthesized in the skin from 7-dehydrocholesterol (7-DHC) by the action of UV-B. There are two principal enzymes involved in the formation of circulating 1,25(OH)2D3 from vitamin D, the hepatic microsomal or mitochondrial vitamin D-25-hydroxylase (CYP27A1) and the renal mitochondrial 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1) for vitamin D and 25(OH)D3 (calcidiol), respectively. Extrarenal activity of CYP27B1 has been reported in various cell types including macrophages, keratinocytes, prostate and colon cancer cells. It has been speculated that the extrarenal CYP27B1-mediated local synthesis of 1,25(OH)2D3 represents a key regulator of important cellular functions including growth and differentiation in various cell types and tissues by autocrine or paracrine signalling pathways. The keratinocyte represents the only cell type where the complete enzymatic machinery for the synthesis of 1,25(OH)2D from 7-DHC is present and where the synthesis of 1,25(OH)2D3 from 7-DHC has been shown. In this study, we characterized the pattern of CYP27B1 splice variants in HaCaT keratinocytes in vitro. Applying nested touch-down PCR, the full length CYP27B1 gene product and several additional CYP27B1 splice variants were detected. The pattern of CYP27B1 splice variants varied depending on the cell density, the calcium concentration of the medium ([Ca2+]o), and UV-B treatment. It can be speculated whether increased expression of CYP27B1 splice variants that lack enzymatic activity (Hyd-V3/V5) may result in a reduction of enzymatic activity and in reduced synthesis of 1,25(OH)2D3. Further study of the impact of CYP27B1 splice variants on the vitamin D pathway in keratinocytes and other cell types is warranted.